A central hypothesis of this proposal is that insulin-dependent diabetes (IDDM) begins with a beta cell targeted attack that unmasks a variety of beta cell antigens leading to the recruitment of distinct population of T-cells that either perpetuate the destructor process or protect against the immune response. According to this view, it is the balance between these opposing influences that determines whether or not IDDM is expressed. The overall goal is to elucidate the molecular mechanisms used by an insulin-reactive cloned CD4T cell line that presents the development of diabetes in NOD mice. this clone, designed 2H6, CD4 produces INFgamma, TNFalpha, as well as TGF beta, but not IL-4, IL-10 or Il-2 i.e., it is a unique "Th1-like" cell. Labeling experiments show that this protective clone trafficks to the islets where it blocks the accumulation of diabetogenic splenocytes with the islet. In vitro experiments indicate that the protective clone or supernatant derived from it inhibits proliferation and the release of IL-2 by a variety of NOD-derived T cells. The specific aims of this proposal are: 1. To examine the mechanisms used by the protective insulin-reactive T cell clone to inhibit the function of diabetogenic T cells. In vitro studies will examine the specificity and nature of the inhibitory activity and its effect on T cell function. The contribution of TGFbeta to the clone's immunoregulatory activity will be examined. In vivo studies will examine the mechanisms used by the protective cells to target the islet, the importance of islet targeting for disease protection, and the capacity of the clone to alter the expression of a variety of cytokines, constimulatory and adhesion molecules after adoptive transfer of (a) diabetogenic splenocytes or () GAD-reactive or insulin-reactive diabetogenic T cells into NOD/SCID mice. We will test the hypothesis that the insulin specific TCR allows the clone to target the islet where it locally releases immunoregulatory factors, specifically TGFbeta that blocks the islet entry and/or activation of diabetogenic T cell. 2. To develop a TCR transgenic NOD mouse derived from the insulin-reactive protective clone and study the natural history of disease expression as well as the biological function of T cells derived from such mice. Alterations in T cell function will be evaluated after the mice are immunized with insulin B chain peptide using a variety of antigenic routes and doses. We will explore modes of activation that promote the appearance of a protective phenotype. These studies should offer insights into the mechanisms for the diabetes protective effects of insulin therapy and could aid in development of a vaccine against the disease.